IFI16 phase separation via multi-phosphorylation drives innate immune signaling

Abstract The interferon inducible protein 16 (IFI16) is a prominent sensor of nuclear pathogenic DNA, initiating innate immune signaling and suppressing viral transcription. However, little is known about mechanisms that initiate IFI16 antiviral functions or its regulation within the host DNA-filled nucleus. Here, we provide in vitro and in vivo evidence to establish that IFI16 undergoes liquid–liquid phase separation (LLPS) nucleated by DNA. IFI16 binding to viral DNA initiates LLPS and induction of cytokines during herpes simplex virus type 1 (HSV-1) infection. Multiple phosphorylation sites within an intrinsically disordered region (IDR) function combinatorially to activate IFI16 LLPS, facilitating filamentation. Regulated by CDK2 and GSK3β, IDR phosphorylation provides a toggle between active and inactive IFI16 and the decoupling of IFI16-mediated cytokine expression from repression of viral transcription. These findings show how IFI16 switch-like phase transitions are achieved with temporal resolution for immune signaling and, more broadly, the multi-layered regulation of nuclear DNA sensors.


INTRODUCTION
Throughout evolution, mammalian cells have developed a complex intrinsic and innate immune system for defense against pathogens by using pattern recognition receptors (PRRs) to detect pa thogen associa ted molecular pa tterns (PAMPs). During viral infection, the nucleic acid that comprises the viral genome is a prototypic PAMP that can be recognized by host PRR sensors of viral DNA or RNA (1)(2)(3). Upon detection and direct binding, these sensors initiate rapid and e xtensi v e signaling cascades that can promote the expression of type I interferons (IFN), pro-inflammatory cytokines, and interferon-stimulated genes. The functions of these signaling molecules during an infection span a broad range of imm une responses, w hich culminate in the intra-and inter-cellular restriction of virus replication and spr ead. To ex ert such functions, a paradigm has emerged supporting the uni v ersal formation of higher-or der PRR assemblies and supramolecular complexes of nanometer to micrometer scales for innate immune signal amplification ( 4 ).
The prevailing hypothesis indicates that IFI16-mediated cytokine induction occurs primarily through signaling to a central cytoplasmic axis, where the endoplasmic reticulum (ER)-resident protein stimulator of interferon genes (STING) activates TANK-binding kinase 1 (TBK-1). TBK-1 subsequently phosphorylates the interferon regulatory factor 3 (IRF3), promoting its dimerization and translocation into the nucleus, where it serves as a transcription factor to induce expression of IFN-stimulated genes and cytokines (21)(22)(23). Despite this kno wledge, ho w IFI16 structur es ar e dynamically r eorganized for immune signal nuclea tion and amplifica tion is less clear and has remained as an acute broader question for PRR sensing and autoimmunity.
We hav e pre viously shown tha t PY-media ted oligomerization is critical for the ability of IFI16 to induce antiviral cytokines and reduce viral spread ( 24 ). The importance of IFI16 oligomerization in promoting its antiviral functions is also substantiated by the existence of multiple virus immune evasion mechanisms tha t inactiva te this property. The HCMV viral tegument protein pUL83 binds IFI16 PY and inhibit its oligomerization ( 25 ), whereas HSV-1 encodes a viral E3 ubiquitin ligase ICP0 that targets IFI16 PY and promotes IFI16 degradation ( 8 , 14 ). A key characteristic of IFI16 oligomerization is its temporally distinct structural states during infection. During early stages of infection, our group and others have observed that IFI16 ra pidl y moves to the incoming viral DNA at the nuclear periphery, followed by a highly dynamic on / off process of puncta formation ( 14 , 26 , 27 ). The second stage is manifested as puncta localizing to nucleoplasmic regions containing nuclear bodies and viral replication compartments ( 14 , 17 , 26 , 28 ). This nuclear localization is followed by the e v entual degradation of IFI16 in the context of wild-type HSV-1 infection ( 8 , 28-30 ). In contrast, when cells are infected with ICP0-null HSV-1 virus and exhibit heightened antiviral cytokine levels, IFI16 oligomerization has been shown to expand into filamentous structures that can recruit host restriction factors to suppress viral replication ( 20 , 29 , 31 ). It was also established that these filaments assembled cooperatively, and the strength of cooperativity is positively correlated with the length of DNA ( 16 ).
Despite such advances, many molecular aspects of IFI16 oligomerization remain unclear. Little is known about the biophysical properties of IFI16 oligomers that govern their morphological changes upon DNA sensing. It is also unclear how IFI16 oligomers are regulated to pre v ent autoimmune activation due to the presence of host DNA. Gi v en that IFI16 cooperati v e binding in vitro was shown to be dependent on DNA length, it has been speculated that the presence of nucleosomes could pre v ent IFI16 diffusion along host DNA and oligomerization ( 17 ), thereby distinguishing self from non-self DNA ( 16 , 31 ). In addition, it remains unknown whether the distinct phases of IFI16, from the dynamic puncta at the nuclear periphery to the stable filaments, have distinct biophysical properties and serve different functions. The mechanisms underlying the transition of IFI16 through these stages of host antiviral response remain to be elucidated.
Here, we provide in vitro and in vivo evidence that the formation of IFI16 puncta and filaments is mediated by liquid-liquid phase separation (LLPS), which is nucleated by interaction with double stranded DN A (dsDN A). Using mutagenesis and microscopy, we have identified an intrinsically disordered region (IDR) on IFI16 that mediates its LLPS. We show that multiple phosphorylation sites within the IDR function in a combinatorial manner to promote IFI16 transition through phase separation stages. Results from mutagenesis and truncation of IFI16 domains lead us to propose a model in which IFI16 filaments are assembled through the end-to-end binding of IFI16 PY and IDR, mediated by PY charged residues and IDR phosphorylations. We further establish that IFI16 LLPS is critical for its induction of antiviral cytokines and inhibition of viral spread, but not for the initial suppression of virus gene expression, providing an example of functional decoupling of the two antiviral functions of IFI16. Additionally, using biochemical enrichment, bioinformatics, and mass spectrometry, we identified CDK2 and GSK3 ␤ as kinases present at the nuclear periphery during infection and contributing to the IFI16 IDR phosphorylation. These observations provide a unifying explanation for how IFI16 switch-like phase separations, from puncta on / off kinetics to filamentation, are achie v ed with high temporal resolution for immune signaling initiation and maintenance. IFI16 oligomerization as dictated by its phosphorylation status serves as a biophysical rheostat that toggles phases of innate immune activity. Overall, our findings uncover biophysical properties of mammalian PRR sensors in the nucleus, pro viding k ey insights into the tight regulation of IFI16 and innate immunity. Tables 1 , 2 and 3 .

Statistical analyses
All PRM data was loaded into and analyzed in Skyline (MacCoss Lab, Uni v ersity of Washington).
All heatma ps, gra phs, and statistical analyses were done in GraphPad Prism 9. Throughout this study, * signifies P ≤ 0.05, ** P ≤ 0.01 and *** P ≤ 0.001, as determined by the significance tests indicated in figure legends. For all mass spectrometry (MS), molecular virology, and microscopy data in this manuscript, the quantification work-  For all infections in this study, cells were grown to 90-100% confluency prior to infection, and inoculations were performed in a quarter of the standard media volume. Cells were infected for 1 h in 2% FBS + DMEM, rinsed in warm PBS, and returned to 10% FBS + DMEM until sample collection. The multiplicity of infection (MOI) varied by experiment, as indicated in the text or figures.

Method details
Plasmid construction. All IFI16-GFP mutant constructs were generated by a site-directed mutagenesis method in the pEGFP-N1 plasmid for transient expression, pBAD plasmid for protein expression and purification, and LentiORF pLEX-MCS for stab le e xpression in HFFs, as described. IDR-IFI16-GFP, FUS-IFI16-GFP and MBP-IFI16-GFP protein constructs were generated using the In-Fusion ® HD Cloning Kit (Takara Bio USA, Inc.) following the manufacturer's instructions.
Plasmids for expression in Ciona embryos were generated using the Sox1 / 2 / 3 regulatory region backbone ( 34 ) IFI16 cassettes were amplified by PCR using proof reading polymer ases (Primestar, Takar a) and assembled using NEB HiFi DNA assemb ly accor ding to the manufacturer's instructions.
Cell line construction, transfections f or e xpression and knockdown, and lentivirus. IFI16 CRISPR-Cas9 KO cell lines were generated as described previously ( 35 ).
For plasmid transfections, HFF cells were seeded at 2 × 10 5 cells / ml the e v ening before transfection with 3 g of DNA and a 1:3 ratio with X-tremeGENE transfection reagent in Opti-MEM. HEK293T cells were seeded at 70% confluency the da y bef ore transfection with 2 g of DNA instead. Cells w ere allow ed to recover f or 24 h bef ore infection.
siRNA oligos wer e pur chased through Sigma Aldrich (custom siRNA consisting of heterogeneous mixture of siRNA that all target the same mRNA sequence). For siRNA-mediated knockdown, HFF cells were seeded at 50,000 to 65,000 cells / ml prior to KD. 20 pmol of siRNA oligos were used per 1 ml well of cells. siRNA was incubated in Lipofectamine RNAiMAX (ThermoFisher Scientific) and Opti-MEM for 5 min, according to the manufactur er's instructions, befor e being added to cells in fresh, complete DMEM. KD cells were left for 48 h before further experiments and were not passaged during siRNA transfections.
RNA isolation and quantitative RT-PCR. Total cellular RNA was purified with the RNeasy Mini kit (Qiagen) following the manufacturer's instructions from 3 × 10 5 HFF cells. Contaminating DNA was digested with DNase I (Invitrogen) for 15 min at room temperature. RNA was re v erse transcribed using the SuperScript IV First-Strand Synthesis Kit (ThermoFisher Scientific) following manufacturer's instructions. Gene-specific primers and the SYBR green PCR master mix (Life Technologies) were used to quantify the resulting cDNA by qPCR on ViiA 7 real-time PCR systems (Applied Biosystems). Relati v e mRNA quantities were determined using the CT method with GAPDH as an internal control.
V irus pr og eny virion titer s. Virus titers were determined by plaque assay on U-2 OS monolayers. Cells were lysed by freezing, and both cell-associated and cell-free viruses were collected, briefly sonicated, and serially diluted. Infections were as above, but after 1 h, the inoculum was replaced with 1% METHOCEL (w / v) in DMEM with 10% FBS. After 72 h, when plaques were evident, the cells were incubated with crystal violet [1% crystal violet (w / v) in 50% methanol (v / v)] for 15 min at room temperature before rinsing and plaque quantification.
MBP-IFI16 was purified in a similar manner and labeled by using Alexa Fluor ™ 488 Protein Labeling Kit (Ther-moFisher) following manufacturer's manual. The estimated degree of labeling was 0.7 mol of Alexa Fluor 488 per mol of IFI16.
In vitro phase separation assay. Assay was performed in Cultur eWell ™ Chamber ed Coverglass (Invitrogen) coated with 20 mg / ml BSA. Mixtures containing indicated components except for MBP-IFI16-GFP were initially added and incubated in 50 mM Tris-HCl pH 8.0, 5% glycerol and 8% Ficoll. Phase separation was achie v ed by adding MBP-IFI16-GFP (in 50 mM Tris-HCl pH 8.0, 500 mM NaCl, 5% glycerol) to the mixtures such that the final concentration of NaCl was 83.3 mM.
In vitro kinase assay. 4 g of purified MBP-IFI16-GFP was incubated with 1 l of indicated purified recombinant kinase in 1X kinase reaction buffer (25 mM Tris-HCl pH 7.5, 5 mM beta-glycerophosphate, 2 mM DTT, 0.1 mM sodium orthovanadate, 10 mM MgCl 2 ), 10 mM ATP and 2 M Cy5-DNA. The samples were incubated at 30 • C with shaking at 300 rpm. The reaction was then stopped by the addition of 4XTES (4% sodium dodecyl sulfate, 20 mM EDTA, 40 mM Tris-HCl pH 7.4) to 1XTES and frozen at −20 • C before sample preparation for mass spectrometry.
In vitro transcription assay. HiScribe TM T7 High Yield RNA Synthesis Kit (NEB) was used following manufacturer's manual. Specifically, 100 ng of FLuC positi v e control template provided by the kit was used for each reaction. 5 M of purified IFI16 or BSA was added to the reaction mix (to a total of 30 l) and incubated at 37 • C for 4 h. 2 l of DNase I (NEB) was added to the reaction mix and incuba ted a t 37 • C for 15 min. Transcribed RNA was isolated by adding 25 l of LiCl solution from the kit, incubated at −20 • C for 30 min and centrifuged at 15,000 × g, 4 • C for 15 min. Pellet was washed with 500 l of 70% ethanol and centrifuged at 15,000 × g, 4 • C for 15 min before being resuspended in 30 l of wa ter. RNA concentra tions were measured using nanodrop.
Fluor escence r ecovery after photob leaching. FRAP was performed using a Nikon A1 confocal microscope equipped with a full incubation chamber maintained at 37 • C and supplied with 5% CO 2 . The point region was bleached for 2 s at 100% of maximum laser power of a 405 nm laser. The recovery was recorded at 100 ms intervals for 2 s, and 1 s intervals for 5 s. Images were analyzed in Fiji. Fluorescence intensities of regions of interest (ROIs) were normalized to pre-bleached intensities of the ROIs. The exponential curve was generated by plotting the normalized fluorescence intensity values to time by GraphPad Prism.
Fluor escence micr oscopy and analysis . For imaging of HFF cells, cells were seeded onto sterile 10.5 × 22 mm glass coverslips (Thermo Fisher Scientific) at 2 × 10 5 cells / ml and infected and treated as indicated. Samples were fixed in 4% paraformaldehyde for 15 min at room temperature and washed three times with PBS. Cells were permeabilized in 0.1% Triton X-100 in PBS for 15 min at room temperature and washed three times with PBS with 0.2% Tween 20 (PBST). Cells were then blocked with 2% bovine serum albumin (BSA), 2.5% human serum in PBST and incubated with primary antibodies for 1 hr (IFI16: Sigma, 1:250; ICP4: Santa Cruz, 1:250 in PBST). Samples were washed three times with PBST and incubated with Alexa Fluorconjugated secondary antibodies (1:2,000 in PBST). Samples were then washed three times with PBST and incubated with 4 ,6-diamidino-2-phenylindole (DAPI) (1:100 in PBS; Thermo Fisher Scientific), washed three times with PBST and mounted using ProLong ™ Diamond Antifade Mountant (Thermo Fisher Scientific).
For imaging of in vitro phase separation, the chambered coverglass was maintained at 30 • C if TEV protease was added using an environmental control chamber, and later lowered to 25 • C after 1 h of incubation.
Imaging experiments were done in the Princeton Confocal Imaging Core using a Nikon A1 confocal microscope or an inverted fluorescence confocal microscope (Nikon Ti-E) equipped with a Yokogawa spinning disc (CSU-21) and digital CMOS camera (Hamamatsu ORCA-Flash TuCam) using a Nikon 100X Plan Apo objecti v e with a 100X magnification or 60X magnification with Nikon 60X Plan Apo objecti v e as indicated. To image cells expressing IFI16-GFP during infection with RFP-tagged VP26 HSV-1, a Nikon Ti-E2 confocal microscopy equipped with a superresolution CSU-W1 SoRa module was used with a Nikon 60X Plan Apo objecti v e. These images were processed with Nikon 3D deconvolution in NIS-Elements AR. Image analysis was performed using ImageJ.
Ciona electr opor ation and imaging. Ciona intestinalis adults were supplied from M-REP (San Diego, CA) and embryos were handled using standard procedures as previously described ( 34 ). Plasmids were electroporated into embryos in cuvettes containing 800 l Mannitol seawater according to standard conditions ( 36 ). 80 g of IFI16-GFP containing plasmids were electroporated and 20 g of H2b::mCh or PH::mAp plasmids were electroporated.
Imaging was performed using a Zeiss 880 Confocal with an Airyscan detector in fast mode using a 63 ×/ 1.40 Planapochromat objecti v e as pre viously described ( 34 ).
Nuclear periphery and core fractionation. The protocol was modified from ( 37 ). About 1.5 × 10 7 HFF cells were used per fractionation. Nuclei of the cells were isolated using NE-PER ™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) following manufacturer's instructions. Isolated nuclei pellets were washed twice with PBS and resuspended in 10 mM Tris-HCl pH 7.4, 1.5 mM KCl, 0.5% Triton X-100, 0.5% Deoxycholate, 2.5 mM MgCl2, with fresh 0.2 M LiCl and protease inhibitors (ratio 1:2 v / v). The mixture was rotated for 1 h at 4 • C and centrifuged at 2,000 × g for 5 min a t 4 • C . Supernatant was further centrifuged at 10,000 × g for 10 min at 4 • C to obtain perinuclear fraction (PNF) from the supernatant. P ellet fraction w as resuspended in 0.34 M sucrose and centrifuged at 2,000 × g for 10 min at 4 • C. The pelleted fraction was dissolved in 8 M urea, sonicated and centrifuged at 10,000 × g for 10 min a t 4 • C . Superna tant from the la test centrifuga tion was collected as the core nuclear fraction (CNF). BCA assay was performed to equilibrate protein concentrations between PNF and CNF prior to sample preparation for mass spectrometry analysis.
TUNEL assay. Each TUNEL experiment included three biological replicates for each condition and the following controls: (i) cells treated with DNase (10 min) for a positi v e control and (ii) cells for a negati v e control (not treated with TUNEL enzyme). A Roche In Situ Cell Death Detection Kit (Sigma Aldrich) was used to detect apoptotic cells in 96-well plates. In short, cells wer e fix ed in 4% paraformaldehyde (PFA) for 10 min at room temperature, washed in PBS, and permeabilized with 0.1% TritonX + 0.1% sodium citrate in PBS for 2 min a t 4 • C . All wells were washed in PBS, and then positi v e control wells were treated with 10 units of DNase I recombinant + 1 mg / ml BSA + 1 M Tris-HCl pH 7.5 in water for 10 min at room temperature. All wells were washed in PBS again, and then incubated for 1 h at 37 • C in TUNEL mix (enzyme solution + label solution). Cells were stained with DAPI and imaged with a Perkin Elmer Operetta automated microscope, scoring apoptotic cells by those positi v e for TUNEL staining.
Immunoaffinity purifications. Cells were washed once with cold PBS and scraped for collection. After pelleting by centrifuga tion a t 300 × g, cells were lysed in lysis buffer ( ples were then frozen at −20 • C or immediately pr epar ed for mass spectrometry analysis. Chr omatin-immunoaffinity purifications . Upon infection of HFF cells with ICP0-RF HSV-1 (2 hpi and MOI of 1), 4 × 10 6 cells per biological r eplicate wer e tr eated with 1% PFA in complete medium (DMEM supplemented with 10% FBS and 1% penicillin / streptomycin) for 7 min at 37 • C. Cold glycine was added to a final concentration of 125 mM and incubated at room temperature for 5 min. Cells were washed twice in cold PBS and scraped in PBS supplemented with 1 × Halt protease and phosphatase inhibitor cocktail (P / PhIC; Thermo Fisher Scientific). Cells were pelleted and lysed in 50 mM HEPES-KOH, 140 mM NaCl, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1 mM EDTA, and 1:100 P / PhIC to isolate nuclei. Nuclei were pelleted upon centrifuga tion a t 1,350 × g for 10 min at 4 • C, subsequently washed in 10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 1:100 P / PhIC, and lysed in 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine, and 1:100 P / PhIC. Samples were then individually sonicated in a cup horn sonicator (20 sec continuously, medium power, repeated 9 times) with 1 min incubation on ice in between rounds. Samples were brought to 1% Triton X-100 and centrifuged at 20,000 × g, for 10 min a t 4 • C . IPs were performed on the soluble fractions using 25 l GFP-Trap MA GFP antibody-coupled magnetic beads (Chromotek) per IP and incubated for 1 h at 4 • C with end-over-end rotation. Beads were washed five times with 50 mM HEPES-KOH (pH 7.6), 100 mM LiCl, 1 mM EDTA, 1% NP-40, and 0.7% sodium deoxycholate, washed once with 10 mM Tris-HCl (pH 8.0), 100 mM EDTA, 50 mM NaCl, and eluted in 50 mM Tris-HCl (pH 8.0), 1% SDS, 10 mM EDTA upon hea ting a t 65 • C in a ThermoMixer (Eppendorf) with shaking at 300 rpm for 30 min. Cross-links were re v ersed in input samples and eluates by incubating at 65 • C overnight. All samples were treated with 0.4 mg / ml RNase A and incuba ted a t 37 • C for 30 min, and 0.4 mg / ml Proteinase K at 55 • C for 2 h. Relati v e le v els of viral genomic DNA were determined by qPCR using the CT method with GAPDH as an internal control.
Sample pr epar ation for mass spectr ometry. Samples were first reduced and alkylated in 5 mM TCEP, 15 mM chloroacetamide for 20 min a t 70 • C , then acidified to 1.2% phosphoric acid prior to protein extraction. Digestion was performed with trypsin (1:25 of trypsin to sample protein) and suspension trapping columns (S-Trap, Protifi) for 1 h a t 47 • C , according to the manufacturer's instructions. Following digestion, peptide eluates were resuspended in 10 l of 1% FA, 1% ACN at a concentration of 1 g / l prior to loading on the instrument.
Mass spectrometry acquisition. For samples collected in PNF / CNF fractionation and IFI16-GFP IP-PRM: Peptides were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry with a Q Exacti v e HF Hybrid Quadrupole-Orbitrap instrument (Thermo Scientific) using data-dependent acquisition (DDA). For DDA, Peptides (2 l injections) were separated with a linear gradient of 3% solvent B to 30% solvent B gradient (solvent A: 0.1% FA, solvent B: 0.1% FA, 97% ACN) over 150 min at a flow rate of 250 nl / min on a self-packed 50 cm Reprosil C18 column (Dr. Maisch GmBH) or with an EASYSpray C18 column (75 m × 50 cm, Thermo) heated to 50 • C. The full scan range was set to 350-1,800 m / z at 120,000 resolution and recorded in profile. The top 15 most intense precursors were subjected to DDA HCD fragmentation with a normalized collision energy (NCE) of 28. Tandem MS / MS spectra wer e acquir ed at a r esolution of 30,000 and an automatic gain control (AGC) target set to 1e5 and a 150 ms maximum injection time (MIT). Precursors were selected with an isolation window of 1.2 m / z . For PRM analyses, peptides were separated chromato gra phicall y identicall y to the DDA method. A full MS survey scan was acquired at resolution of 30,000, an AGC target of 3e6, MIT of 15 ms, scan range of 350-1,800 m / z , and data was collected in profile. For the PRM scans, the resolution was set to 30,000, an AGC target of 1e5, a MIT of 60 ms, loop count of 20, pr ecursors wer e selected with a 0.8 m / z isolation window, a fixed first mass of 125 m / z , NCE of 27, and spectra were collected in profile.
For samples collected in in vitro kinase assays: Peptides from samples with kinase added were pooled to make a master mix and subjected to DDA analysis. Peptides (2 l injections) were separated with a linear gradient of 3% solvent B to 30% solvent B gradient (solvent A: 0.1% FA, solvent B: 0.1% FA, 97% ACN) over 60 min at a flow rate of 250 nl / min on a self packed 50 cm Reprosil C18 column (Dr Maisch GmBH) heated to 50 • C. The full scan range was set to 350-1500 m / z at 120,000 resolution and recorded in profile. The top 20 most intense precursors were subjected to DDA HCD fragmentation with a normalized collision energy (NCE) of 28. Tandem MS / MS spectra wer e acquir ed at a r esolution of 15,000 and an automatic gain control (AGC) target set to 1e5 and a 25 ms maximum injection time (MIT). Precursors were selected with an isolation window of 1.2 m / z . For PRM analyses, peptides were separated chromato gra phicall y identicall y to the DDA method. A full MS survey scan was acquir ed at r esolution of 120,000, an AGC target of 3e6, MIT of 200 ms, scan range of 150-2,000 m / z , and data was collected in profile. For the PRM scans, the resolution was set to 15,000, an AGC target of 2e5, a MIT of 200 ms, loop count of 25, pr ecursors wer e selected with a 1.2 m / z isolation window, a fixed first mass of 125 m / z , NCE of 27, and spectra were collected in profile.
PRM library and analysis of MS data. PRM assays were designed and analyzed using the Skyline Daily software. Dominant proteotypic peptides for each IFI16 phosphorylation site were selected based on detection le v els in DDA and the elution and fragmentation profiles were experimentally determined for each targeted peptide. Peptide abundance was quantified using the summed area under the curve of 3-5 fragment ions per peptide. Peptide abundance values were normalized by the abundance value of a selected unmodifiable peptide of IFI16 and scaled to the mock condition in each IP. Two-tailed Student's t -tests were performed using GraphPad Prism 9.
Tandem MS spectra collected from DDA mode were analyzed by Proteome Discoverer v2.4 (Thermo Fisher Scientific). For identification of protein enriched in PNF, abundance ratio for each protein was calculated by taking the ratio of PNF / CNF of average abundance values across biological replicates, and two-tailed Student's t -tests were performed to calculate the P -value for the abundance ratio.

IFI16 undergoes liquid-liquid phase separation in vitro
We pre viously observ ed tha t IFI16 undergoes d ynamic changes in its subnuclear localiza tion a t the early stages (30 min-2 h) of HSV-1 and HCMV infections ( 14 ). IFI16 leaves the nucleolus and forms discrete foci at sites of viral genome deposition at the nuclear periphery ( 14 ). The IFI16 foci have a dynamic behavior, forming round puncta that are uniform in size and that appear and disappear on the order of minutes (Supplementary Figure S1A, Movie 1). The initial enrichment of IFI16 within nucleoli, which are phaseseparated organelles, and its dynamic spherical puncta formation led us to ask whether IFI16 undergoes liquid-liquid phase separation (LLPS) during the initial stages of binding to incoming viral DNA. LLPS is a process involving the formation of membrane-less compartments mediated by weak interactions among the components, and has been shown to be involved in regulating intracellular organization, signaling tr ansduction, tr anscription, and cell str ess r esponse (38)(39)(40)(41).
We first determined whether IFI16 alone is sufficient to cluster DNA in vitro (Figure 1 A). Purified IFI16 (10 M) was mixed with equimolar amounts of Cy5-labelled vaccinia virus dsDNA 70mer (VACV 70mer). Cyclic GMP-AMP synthase (cGAS), a DNA sensor known to undergo LLPS in the cytoplasm ( 41 ), was included as a positi v e contr ol. Confocal micr oscopy showed r obust Cy5-DNA puncta formation in the presence of either IFI16 or cGAS (Figure  1 B), suggesting that either IFI16 or cGAS is sufficient to cluster DNA in vitr o . As nega ti v e controls, we incubated Cy5-DNA with either BSA, which does not bind to DNA, or DN A-PK, w hich binds to DN A but has not been shown to undergo LLPS. Both proteins failed to induce Cy5-DNA puncta (Figure 1 B). Although we did not observe a difference in the size of the Cy5-DNA puncta in the presence of cGAS or IFI16, we noticed that DNA puncta induced by cGAS appeared as more spherical than those induced by IFI16 (Figure 1 B). This may be dri v en by the difference in the type of oligomeriza tion tha t cGAS or IFI16 undergo, with cGAS known to dimerize and IFI16 shown to undergo oligomeriza tion tha t can expand into filaments.
To directly visualize IFI16 morphology upon its binding to DNA in vitro , we purified IFI16-GFP with a maltose binding protein (MBP) and 3X TEV protease cleava ge sites ta gged at the IFI16 N-terminus (Figure 1 C). Only in the presence of DNA, MBP-cleaved IFI16-GFP formed micr ometer-sized spherical dr oplets that colocalized with Cy5-DNA (Figure 1 D). The IFI16 droplet formation was concentration-dependent, being most apparent at the lowest concentration tested, i.e. 1 M. At a higher concentration (3 M), in addition to forming droplets, IFI16-GFP started to expand into filamentous structures (Fig-ure 1 D). The prevalence of droplet formation at 1 M was confirmed by quantification (Figure 1 E). Further increasing the IFI16 concentration to 10 M caused the MBPcleaved IFI16-GFP to display fully filamentous structures (Figure 1 F, first row). Additionally, e v en at 10 M, the presence of MBP, which has been reported as a solubilityenhancing tag ( 42 , 43 ), in the uncleaved MBP-IFI16-GFP led to droplet formation that colocalized with Cy5-DNA (Figure 1 F, second row and Movie 2). The observed state for the uncleaved IFI16 fits into a model in which the MBP affects the interaction strength among IFI16-GFP molecules, likely by inhibiting PY-dependent homotypic interactions and IFI16 oligomerization. Altogether, these results illustra te tha t the clustering of IFI16-GFP is dependent on the presence of DNA, and its ma terial sta te transition from droplets to filaments is linked to its concentration and interaction strength, fitting into the canonical model for proteins capable of undergoing LLPS (Figure 1 G) ( 44 ).
To further validate the IFI16 LLPS in vitro , we incubated low or high concentrations of IFI16-GFP (1 or 20 M) with Cy5-DNA in the presence or absence of 1,6-hexanediol (1,6-HD), a chemical reported to selecti v ely inhibit liquid-phase condensates ( 45 ). For both concentrations tested, IFI16-GFP morpholo gy was significantl y altered by the addition of 1,6-HD (Figure 1 H-I). At 20 M, the presence of 1,6-HD, which was added prior to the addition of the TEV protease and the purified IFI16 protein, pre v ented filament formation, arresting IFI16-GFP in a droplet form (Figure 1 H). The observa tion tha t filaments are not completely dissolved by 1,6-HD could be attributed to a number of factors, including the primary inhibition of hydrophobic interactions by this treatment ( 45 ) and expected maintenance of IFI16-DNA interaction and PY charge-media ted oligomeriza tion. Under the same condition at 1 M, the IFI16-GFP droplets were abolished (Supplementary Figure S1A). Further supporting the involvement of LLPS in IFI16 droplet formation, we observed that droplets fused ra pidl y, within seconds (Figure 1 J). Altogether, the in vitro spherical puncta formation in the presence of DNA, together with the response to 1,6-HD treatment and the observed droplet fusion all point to the ability of IFI16 to undergo LLPS in vitro .
To confirm that the C-terminal GFP did not impact the IFI16 LLPS capacity, we purified untagged IFI16, which we then labeled with Alexa Fluor 488. Similar to IFI16-GFP, untagged IFI16 displayed a concentration-dependent LLPS morphology, forming droplets and filaments at similar concentrations (1 and 10 M respecti v ely, Figure 1 I and S1B). The droplets formed by untagged IFI16 were also inhibited by 1,6-HD (Figure 1 I). These data suggest that the GFP did not affect the LLPS capacity of IFI16 in vitro .
We next asked whether IFI16 also displays LLPS behavior in cells. Within the nucleus, IFI16 has been shown to display nucleolar, as well as diffuse nuclear localizations, which may be linked to different cell states and cell types, as well as different epitopes targeted by the antibodies used. To assess a possible IFI16 LLPS behavior in cells, we first monitored uninfected cells where we noticed pronounced nucleolar localization. Using primary human fibroblasts stab ly e xpressing IFI16 fused to monomeric eGFP (IFI16-eGFP), we monitored recovery after photobleaching within the nucleolus. IFI16 fluorescent signals r ecover ed ra pidl y,  within seconds, suggesting that nucleolar IFI16 exhibits a highly dynamic exchange with its surroundings (Figure 2 A-B). The nucleolar population of IFI16 likely contributes to the formation of perinuclear puncta during infection (Supplementary Figure S1C), as we observed an anti-correlation between the fluorescence intensities of IFI16-eGFP in the nucleolus and those at the nuclear periphery upon infection with HSV-1 (Supplementary Figure S1D). To assess whether LLPS is involved in the recruitment of IFI16 to in-coming viral DNA, we tested the impact of 1,6-HD treatment on IFI16 localization during HSV-1 infection. As HSV-1 is known to target IFI16 for degradation ( 8 ), we performed infections with an HSV-1 strain that harbors a mutation in the ring finger (RF) domain of the viral E3 ubiquitin ligase ICP0 and thereby lacks the ability to suppress IFI16 functions ( ICP0-RF HSV-1). As expected, we observed IFI16-GFP puncta localizing at the nuclear periphery, at sites of viral genome deposition, early in infection, i.e. at 1 h post infection (hpi) with ICP0-RF HSV-1 (Figure 2 C). By 8 hpi, IFI16-GFP displayed a filamentous appearance (Supplementary Figure S1E). In both instances, IFI16 co-localized with the vir al immediate-ear ly protein ICP4, which marks sites of viral genomes. These findings wer e r eca pitulated w hen staining for endo genous IFI16 at similar time points, indicating that these phenotypes are not an artifact of the ov ere xpression system (Figure 2 D). Treatment with 1,6-HD inhibited the ability of either IFI16-GFP or endogenous IFI16 to become enriched and form puncta at viral genome sites at the nuclear periphery (Figure 2 C-D). On the other hand, as expected when considering the metastability of proteins with LLPS properties ( 44 ) (Figure 1 G), once IFI16 filaments were formed at 8 hpi, 1,6-HD treatment was no longer able to re v erse this solid-like state (Figure 2 D). These observations confirm that the 1,6-HD treatment specifically inhibits liquid-state condensates. Taken together, these results demonstrate that the puncta formation of IFI16 is mediated by LLPS, and that this condensation ability is r equir ed for the localization of IFI16 to viral genomes at the nuclear periphery.

IFI16 contains an intrinsically disor der ed r egion r equir ed f or LLPS and antiviral function
Proteins with capabilities to undergo LLPS often contain multivalent domains or intrinsically disordered regions (IDR) to mediate weak and tr ansient inter actions ( 44 ). To determine if IFI16 contains disordered regions, we performed a bioinformatics analysis using Prediction of Intrinsically Unstructured Proteins (IUPred3) ( 46 ). Two regions of disorder wer e pr edicted (aa 88-188 and 347-574, Figure  3 A). The most evident IDR region, positioned between the PY and first HIN domain, coincides with the region that we pr eviously r eported to be decorated by multiple phosphorylation sites ( 11 , 35 ). Accumulating evidences have suggested that phosphorylation e v ents within disordered regions can contribute to either promoting or suppressing LLPS ( 47 , 48 ), with a recent stud y demonstra ting phosphoryla tion signa tur es pr esent specifically in either condensates or the soluble populations of phase separating proteins ( 49 ). Hence, we focused on characterizing the possible contribution of the first IDR to the observed IFI16 LLPS. We generated IFI16-GFP constructs containing either an IDR deletion ( IDR-IFI16) or a replacement of the IFI16 IDR with FUS aa 1-163 (FUS-IFI16) (Figure 3 B), an IDR known to be sufficient to induce LLPS ( 42 ). Following the equivalent expression of these IFI16-GFP constructs in primary human fibroblasts (HFFs) and HEK293T cells (Supplementary Figure S2A), we quantified the percentage of cells displaying diffuse or LLPS IFI16-GFP phenotypes, either punctate or filamentous (Supplementary Figure S2B). We found that IDR-IFI16 lost the ability to form puncta or filaments, e v en in cells with high expression levels of this construct, whereas the FUS-IFI16 rescued this ability to the WT le v el (Figure 3 C-E). We next asked if the loss of LLPS impacts the antiviral function of IFI16. For this, we used an IFI16-deficient cell line that expresses STING (HEK293T-STING) and which has been shown to allow the reconstitution of the STING-TBK1-IRF3 signaling axis to assess cellular antiviral responses ( 14 , 50 ). Infection of cells expressing IDR-IFI16 with ICP0-RF HSV-1 resulted in three-fold higher virus titers than cells expressing WT-IFI16 and greater than two-fold higher than cells expressing FUS-IFI16, indicating a defect in the ability of IDR-IFI16 to inhibit viral production (Figure 3 F). Altogether, these results suggest that the IDR is necessary for promoting IFI16 LLPS and antiviral function.

IFI16 IDR regulates LLPS in vivo
To determine whether the IDR is also relevant in regulating IFI16 aggregation in vivo, we took advantage of a recently de v eloped system for observing nuclear proteins in the embryos of Ciona intestinalis ( 34 ). Ciona di v erged from modern v ertebrates ov er 500 million years ago and provides an evolutionarily simple model system ( 51 , 52 ). Directly relevant for this study, we selected Ciona given reports that demonstrated its ease of use specifically for visualizing the dynamics of phase separating proteins with high spatial and temporal resolution as they transition across different biophysical states ( 34 ). Fluorescently-tagged proteins can be r eadily expr essed by electroporation in Ciona embryos, and the large nuclei of these embryo enhance the visualization of condensates for nuclear proteins. As Ciona does not express a direct sequence homolog of IFI16, it provides the ability to reconstitute and monitor IFI16 material states in an in vivo model system. We expressed WT, IDR, or FUS IFI16-GFP in the ectoderm of li v e, de v eloping Ciona embryos under the regulation of a Sox1 / 2 / 3 promoter (Figure 3 G). The concentration-dependent distributions and dynamics of these IFI16 constructs were visualized in li v e embryos using time-lapse microscopy (Movie 3). WT IFI16-GFP displayed evident nuclear localization in early gastrula embry os, being distributed unif ormly throughout the nucleus (Figure 3 H). No IFI16-GFP was retained on the chromatin during mitosis. After the first mitosis, puncta formed, typically starting at the sites closest to the former cleavage furrow. By the mid gastrula stage, IFI16 puncta were consistently visible at the nuclear periphery ( Figure 3 H and I).
These puncta grew into larger nuclear aggregates by the late gastrula stage (a pproximatel y two hours and two rounds of mitosis later, Figure 3 J). After these aggregates formed, IFI16 was retained on the chromatin during the next mitosis. In contrast, IDR IFI16-GFP retained a diffuse nuclear distribution throughout gastrula stages ( Figure 3 J and Supplementary Figure S2C), with no aggregation being detected e v en a t la te stages (Figure 3 J). Similar to our observations in mammalian cells, the addition of a FUS domain rescued the ability of IFI16 to a ggregate. FUS-IFI16-GFP a ggr egates wer e evident immedia tely, starting a t early gastrula stages (Supplementary Figure S2C). The FUS-IFI16-GFP fibrous aggregates were retained on the chromatin after the first mitosis and were present throughout de v elopment (Supplementary Figure S2C, Movie 4). These aggregates had a filamentous appearance, forming multiple string-like patterns. Since e xpression le v els of transgenes in Ciona embryos can be variable, we took advantage of this property and measured the fluorescence intensity of IFI16-GFP in neurula stage embryos, comparing nuclei where aggregates were observed with those without aggregates. Nuclei that contained aggregates typically had 2-to 3-fold higher fluor escence intensities (Figur e 3 K), suggesting that a critical nuclear concentration of IFI16 needs to be reached for aggregates to form. Altogether, consistent with our findings in mammalian cell culture, these results demonstrated that the WT IFI16 forms concentration-dependent puncta and filamentous aggregates in vivo , a property that r equir es the presence of the IDR.

Multiple phosphorylation sites within the IDR combinatorially regulate IFI16 LLPS
Having established the importance of the IDR in regulating IFI16 LLPS in vitro and in vivo , we next asked what properties of the IDR facilitate this function. Gi v en that we pre viously observ ed that this IFI16 region contains multiple phosphorylation sites ( 11 , 35 ), we asked whether these phosphorylation e v ents (S95, S106, T149, S153, S168, and S174) contribute to the regulation of the IFI16 LLPS. Using sequence alignment, we observed that most of the identified phosphorylation sites within the IDR are conserved among dif ferent prima te species (Supplementary Figure S3A, B). Next, we assessed whether these IFI16 sites become phosphorylated early in HSV-1 infection, at time points when IFI16 binds to the incoming viral DNA and exerts the dynamic puncta behavior. To accurately define the IFI16 phosphoryla tion sta tus, we designed a targeted mass spectrometry assay using parallel reaction monitoring (PRM) that detects signature parameters for each of the predicted phosphorylated peptides. This PRM assay was then applied following enrichment of IFI16 via immunoaffinity purification (IPs) at one-and six-hours post infection (hpi) with ICP0-RF HSV-1 (Supplementary Figure S4A, Supplementary Table S1). We reliably detected fragment ion peaks for fiv e out of the six candidate phosphorylated peptides (Supplementary Figure S4B), confirming the presence of S95, S106, T149, S153 and S168 phosphorylation sites at both one and six hpi (Supplementary Figure S4C).
To determine whether the identified phosphorylation sites impact the IFI16 LLPS capability, we generated a series of single phosphorylation mutants. Each site was individually converted from a serine / threonine to either an alanine or aspartate to introduce an unmodified residue or a phosphorylation mimic, respecti v ely (Figure 4 A). In agreement with our in vivo observations, filamentous IFI16 phenotypes wer e corr elated with IFI16 expr ession le v els in cells (Supplementary Figure S4D). Howe v er, the presence of the single phospho-mutants did not significantly change the ability of IFI16 to form puncta or filaments (Supplementary Figure S4E-F). Additionally, the expression of equivalent le v els of the single mutants (Supplementary Figure S4G) did not result in significant differences in virus titers when compared to the WT IFI16 (Supplementary Figure S4H). Hence, we reasoned that a single phosphorylation e v ent is not sufficient to control the LLPS status or antiviral function of IFI16.
To assess whether a combinatorial effect is at play, we generated IFI16 constructs containing all six phosphoryla ted residues muta ted to alanine or asparta te (6A-and 6D-IFI16-GFP, respecti v ely) and e xpressed these in HFFs (Figure 4 A). In the absence of infection, 6A-IFI16-GFP m utant transientl y expressed in HFFs lost the ability to form puncta or filaments, whereas the 6D-IFI16-GFP displayed normal or e v en enhanced puncta and filament formation compared to WT IFI16 (Figure 4 B-C). Gi v en these differences, we next investigated these combinatorial phosphomutants during infection. To ensure that the tested impact on IFI16 localization and antiviral functions is the result of the presence of the phosphomutants and not contributed by endogenous IFI16, we generated IFI16 CRISPR / Cas9-mediated knockouts in HFFs and stably expr essed CRISPR-r esistant forms of WT-IFI16-GFP, 6A-IFI16-GFP or 6D-IFI16-GFP in the knockout background (Supplementary Figure S4I).
Using the stable cell lines expressing the phosphomutants, we examined the impact of phosphorylation on the ability of IFI16 to bind incoming viral genomes at the nuclear periphery during the initial stages of HSV-1 infection. To be able to track virus capsids, we infected the stable cell lines with an HSV-1 strain that expresses the major capsid surface protein, VP26, fused to monomeric red fluorescent protein ( HSV-1::rfp-vp26 ). Upon detection of RFP-tagged viral capsids at the outer nuclear periphery, we observed the simultaneous and adjacent association of WT, 6A or 6D IFI16-GFP puncta at the inner nuclear periphery during early infection with ICP0-RF HSV-1 at 2 hpi (Supplementary Figure S5A). As an orthogonal approach to confirm that phosphorylation does not impact the capacity of IFI16 to bind viral DNA during infection, we performed a chroma tin immunoprecipita tion (ChIP) followed by quantitati v e PCR at 2 hpi, when viral genomes enter the nucleus. We found that both 6A and 6D IFI16 are capable of binding to viral genomes to a similar extent as WT IFI16 (at genomic loci ul30 and ul5 , Supplementary Figure S5B). Altogether, these findings indicate that the phosphorylation state of IFI16 does not impact the ability of IFI16 to be recruited to the site of viral genome deposition and bind to viral DNA.
We next investigated whether the localization of IFI16 to viral genomes is sustained throughout infection upon the de v elopment of nuclear viral replication compartments. We analyzed cells within de v eloping HSV-1 plaques and assessed IFI16 co-localization with a viral protein marker for viral genomes, ICP4, at both plaque edges (early infection) and plaque interiors (late infection). Early in infection, we observed that both 6A and 6D mutants of IFI16 retain their co-localization with ICP4 at the nuclear periphery. In contrast, later in infection, relati v e to either WTor 6D-IFI16 formation of filaments within viral replication compartments, 6A-IFI16 failed to transition from initial puncta to filaments (Figure 4 D). These observations suggest that the phosphorylation state of IFI16 impedes its ability to progress through LLPS stages (Figure 1 G) to filaments.
Our observa tions tha t 6A-IFI16 still forms puncta a t the nuclear periphery early in infection, but fails to de v elop filaments later in infection, led us to propose that the initial puncta formation at the nuclear periphery is facilitated by DNA-binding and PY-mediated oligomerization. These puncta then transition through IDR phosphorylationregulated phase separation stages as viral replication compartments form, transitioning from droplet-like aggregates to filaments at later stages of infection. To test this hypothesis, we generated IFI16 truncation mutants bearing 1) only the PY domain (PY-GFP), 2) only the IDR domain (IDR-3XFLAG), and 3) both PY and IDR domains (PY-IDR-GFP, as in ( 14 )) (Figure 4 E). We observed robust puncta f ormation f or PY-onl y m utants, suggesting that the PY domain alone is sufficient to gi v e rise to puncta. Howe v er, filamentation was only observed in cells expressing PY-IDR-GFP (Figure 4 F). These results suggest that both PY and IDR domains are required for IFI16 filamentation, implying a possible synergistic interaction between the two domains.
To investigate the relationship between the IDR LLPS and PY oligomerization functions, we took advantage of our previously identified point mutations that can control the IFI16 PY-mediated oligomerization ( 24 ). Specificall y, m utating the PY R23 residue to a structural mimic (R23Q), but not to a charge mimic (R23K), inhibited IFI16 oligomeriza tion. Hence, we genera ted combina torial IFI16 mutants bearing gain-or loss-of-function mutations within both PY and IDR and tested their abilities to form aggregates in cells by confocal microscop y (Figur e 4 G). Cells transiently expressing IFI16 that is deficient in either oligomerization or LLPS failed to form filaments. In contrast, IFI16 bearing gain-of-function mutations in both PY and IDR (R / K-6D) rescued the ability to form filaments (Figure 4 H-I). This indica tes tha t the oligomerization and LLPS abilities of IFI16 act synergistically to promote its aggrega te forma tion in cells, which is necessary for its antiviral functions.

IFI16 LLPS-deficient mutant has dampened capacity for cytokine induction but maintained ability to suppress viral gene expression
Having established a combinatorial role for IFI16 phosphorylations in promoting transition through phase separation stages, we sought to determine their effect on the antiviral functions of IFI16 in either inducing cytokine expression or suppressing viral gene expression. To examine the impact of phosphorylation on viral gene suppression, we infected IFI16 KO, WT, 6A or 6D stable cell lines with ICP0-RF HSV-1 and used quantitati v e re v erse transcription PCR (RT-qPCR) to measure the mRNA le v els of HSV-1 immediate-early (IE, icp4 ) and early (E, icp8 ) genes at 6 hpi. No major differences in the expression of these IE and E genes were observed in cells expressing either WT or mutant forms of IFI16 at 6 hpi ( Figure 5 A). To further confirm the retained transcriptional suppression capabilities of the IFI16 phosphomutants, we performed an in vitro transcription assay, as in ( 53 ). For this assay, we first purified WT-, 6A-and 6D-IFI16-GFP, which we then co-incubated with an in vitro transcription reaction using a linearized plasmid containing the firefly luciferase gene (Fluc) under the transcriptional control of T7 promoter. In the presence of WT IFI16, RNA pr oduced fr om transcription was reduced by ten-fold, indica ting tha t IFI16 is suf ficient to suppress transcription in vitro . This transcriptional suppression ability was inhibited in the presence of excess non-template doublestranded DNA (Figure 5 B), in agreement with previous reports that IFI16 can bind indiscriminately to DNA in vitro ( 15 , 54 ). Importantly, both 6A-and 6D-IFI16 were equally capable of suppressing transcription in vitro , to an extent similar to the WT, whereas BSA, as a negati v e control, did not inhibit transcription at the same concentration. These da ta indica te tha t IFI16 is suf ficient to suppress transcription and that this ability is independent of its phosphorylation state.
We then asked if the capacity of IFI16 to induce cytokine expression was impacted by its phosphoryla tion sta te. Using the stable cell lines described above, we performed qPCR to measure the mRNA le v els of cytokines ( isg54 and isg56 ) during ICP0-RF HSV-1 infection at 6 hpi. We found that cells expressing 6D-IFI16 induced similar le v els of cytokines compared to those expressing WT-IFI16. In contrast, cells expressing 6A-IFI16 had dampened cytokine expr ession (Figur e 5 C). Consistent with this finding, infected 6A-IFI16 expressing cells had diminished ability to limit viral spread, as seen by the higher virus titer when compared to cells expressing WT or 6D IFI16 at 24 hpi (i.e. after at least one round of virus replication), a phenotype further enhanced at 48 hpi (i.e. after multiple rounds of replication) (Figure 5 D).
Overall, these results indica te tha t IDR phosphorylation does not impact the ability of IFI16 to suppress transcription either in vitro or in cells during infection, and that phosphorylation-mediated phase separation of IFI16 specifically promotes cytokine induction.

CDK2 and GSK3 ␤ directly phosphorylate the IFI16 IDR
Having determined a role for combinatorial IDR phosphorylation for IFI16 LLPS and antiviral functions, we next asked which kinases target the IFI16 IDR for phosphoryla tion. We hypothesized tha t these kinases would localize to the nuclear periphery early in infection. Additionally, such a combinatorial modification status would result either from the action of one kinase phosphorylating multiple sites or of multiple kinases modifying distinct sites. To identify infection-and localization-dependent kinases, we designed a two-pronged approach, integrating biochemical fractionation with computational kinase prediction. Specifically, at 1 hpi with ICP0-RF HSV-1, when IFI16 perinuclear puncta form, we separated the nuclear periphery from the nuclear core into distinct fractions (Figure 6 A). The effecti v eness of this fractionation protocol was confirmed by monitoring the enrichment of nucleoporins and nuclear lamina proteins in the perinuclear fraction (PNF) and of histones and nucleolar proteins in the core nuclear fraction (CNF) using western blot and quantitati v e mass spectrometry ( Figure 6B, S6A and Supplementary Tab le S2). Ne xt, using mass spectrometry analysis, we identified 34 kinases specifically enriched at the nuclear periphery when compared to the nuclear core fraction at 1 hpi (Figure 6 C). These PNF-enriched kinases were further probed using se v eral kinase prediction tools that analyze sequence motifs, focusing on the IDR phosphorylation sites that we tested via mutations (Figure 6 A). Overall, 12 kinases passed the two criteria of being enriched in the PNF and bioinformatically predicted as possibly targeting the IFI16 IDR sites (Figure 6 D, left). Among these, se v eral cy clin-dependent kinases (particularly CDK2) and the gl yco gen synthase kinase 3 beta (GSK3 ␤) were the top candidates ( Figure 6 D, right). Both of these kinases were predicted to phosphorylate multiple IFI16 sites (Supplementary Figure S6B). To experimentally confirm their activity on the IFI16 IDR, we used an in vitro kinase assay and designed a targeted mass spectrometry method for the detection and quantification of the IDR phosphorylated peptides. In addition to these top two kinase candidates (CDK2 and GSK3 ␤), we tested the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs, or PRKDC), which we have pr eviously r eported to phosphorylate IFI16 at T149 ( 35 ) and our current bioinformatics analysis also predicted to act on an additional IDR site (Supplementary Figure S6B). Specifically, we incubated purified IFI16-GFP and DNA with the acti v e form of each purified kinase in the presence or absence of its specific inhibitor. We then subjected the samples to targeted mass spectrometry to relati v ely quantify the phosphorylated IFI16 IDR peptides. Fi v e of the IDR sites --S95, S106, T149, S153 and S168 --were found to be phosphorylated in a kinase activity-dependent manner (Figure 6 E, Supplementary Table S3). In agreement with our prior report and our curr ent bioinformatics pr ediction, DNA-PK phosphorylated the T149 site, acting as a positi v e control for our in vitro assay. Additionally, we discovered that GSK3 ␤ phosphorylates the IFI16 S95 site, an activity diminished by treatment with a GSK3 ␤-specific inhibitor, TWS-119. CDK2 was found to modify multiple sites, S106, S153 and S168, an activity reduced upon treatment with a specific inhibitor, CDK2-IN-4.
To determine whether the kinase activities we observed in vitro support IFI16-mediated cytokine response, we infected IFI16-KO HFFs or scrambled-KO HFFs with ICP0-RF HSV-1 and treated the cells with vehicle control or AT7519, a pan-CDK inhibitor that also inhibits GSK3 ␤ ( 55 ). Upon verifying that the inhibitor treatment does not promote apoptosis (Figure 7 A), we measured the mRNA abundance of interferon-stimulated genes, isg54 and isg56, by RT-qPCR. In the control cells (scrambled-KO HFFs), kinase inhibition led to reduced isg54 and isg56 expression le v els (Figure 7 B). This effect was absent in IFI16-KO HFFs, indica ting tha t the ef fect of the kinase modulation on cytokine induction is dependent on IFI16. Gi v en our finding that CDK2 phosphorylates multiple IFI16 sites, we next specifically examined the role of CDK2 in regulating IFI16 LLPS and IFI16-dependent cytokine induction in cells. Upon siRNA knockdown of CDK2 (Figure 7 C) in IFI16-GFP e xpressing HFFs, we observ ed a significant reduction in IFI16 filament formation during infection (Figure 7 D, E). Additionally, CDK2 knockdown also led to reduced isg54 and isg56 le v els in scramb led-KO HFFs, but not in IFI16-KO HFFs during infection (Figure 7 F). Altogether, our results demonstrate that CDK2 is present at the nuclear periphery early in HSV-1 infection and induces the phosphorylation of multiple sites within the IFI16 IDR, ther eby r egulating the induction of cytokine expr ession.

DISCUSSION
When facing foreign insults, cells must maintain a delicate balance that allows for an effecti v e immune defense mechanism, while ensuring that the response is not too strong to cause harmful autoimmunity. This is especially critical in the context of nuclear DNA sensing. The nucleus is full of host chromosomes, so the DNA sensors need to distinguish between foreign and host DNA to avoid aberrant spontaneous activation, and at the same time also be tightly regulated to activate specifically in the presence of foreign DNA. For example, aberrant expression of the nuclear DNA sensor IFI16 has been associated with several autoimmune diseases, including Sj ögren's syndrome ( 56 ), systemic lupus erythematosus ( 57 ), systemic sclerosis ( 58 , 59 ), inflammatory bowel disease ( 60 , 61 ), rheumatoid arthritis ( 58 ), and psoriasis ( 62 , 63 ). It has also been suggested that IFI16 filamentation with DNA contributes to its autoantigen status in Sj ögren's syndrome ( 64 ). Thus, investigations into the mechanistic cues that toggle DNAdependent IFI16 oligomerization during contexts of PRR sensing have important implications not only for understanding antiviral immunity, but also for autoimmune diseases and imm unothera pies.
Here, we establish that the innate immune PRR sensor IFI16 undergoes LLPS in the presence of dsDNA invitro and in vivo. We discovered an intrinsically disordered region on IFI16 that regulates its LLPS capability, and demonstra ted tha t CDK2 acts on multiple sites within the IDR to promote LLPS and cytokine induction (Figure 7 G).
In vitro, we demonstrate that IFI16 LLPS controls the formation of two distinct biophysical modalities, droplets and . Perinuclear fold enrichment for each kinase was calculated as the grouped abundance value of perinuclear fraction over that of core nuclear fraction across three biological replicates. Frequency of prediction by bioinformatics was plotted for kinases with statistically significant ( P -value < 0.05) perinuclear fold enrichment. ( E ) Normalized abundances for indicated phospho-peptides measured by quantitati v e mass spectrometry. 4 g of purified MBP-IFI16-GFP were incubated with Cy5-DNA and 1 l indicated purified acti v e kinase in the presence or absence of its corresponding inhibitor for 1 h. Samples were then quantified by parallel reaction monitoring (PRM). Values are means ± SEMs ( n = 3). Samples with corresponding inhibitors added were indicated with 'i' appended at the end of the kinase name. Statistical analysis was performed using ordinary one-way ANOVA. Relati v e mRNA le v els (means ± SEMs, n = 3) of isg54 and isg56 in IFI16-KO HFF stable cell lines or scrambled-KO HFFs infected with ICP0-RF HSV-1 (MOI = 5) after negati v e control or CDK2 knockdown, measured by RT-qPCR at 6 hpi. Statistical analysis was performed using unpair ed t -test. Values ar e means ± SEMs ( n = 3). ( G ) Model for IFI16 LLPS in immune activation. IFI16 molecules first bind to viral DNA at the nuclear periphery, giving rise to puncta. CDK2 phosphorylates IFI16 IDR at S106, S153 and S168, facilitating LLPS of IFI16. As infection progresses and IFI16 expression increases, IFI16 transitions from a puncta te sta te to solid filamentous state, which is mediated by interactions between positi v ely charged residues within PY and phosphorylated residues within IDR. Finally, LLPS of IFI16 activates downstream signaling pathways to promote cytokine induction. these findings, we propose a model in which IFI16 filamentation is dri v en by multivalent interactions between PY and IDR, thus allowing for a mesh-like binding of IFI16 that gi v es rise to filaments (Figure 7 G).
Taken together, our findings point to LLPS as a key biophysical pr er equisite to initiate IFI16 antiviral functions. The r equir ement for a thr eshold in both concentration and phosphorylation le v els enab les a switch-like behavior for the activation of IFI16 to carry out its antiviral functions. These findings also have important mechanistic implications for mammalian PY-containing proteins with potential IDRs immediately C-terminal of the PY, most of which have reported roles in modulating immunity (e.g. NLRPs, ASC, and PYHIN proteins AIM2, IFIX) ( 5 , 74-78 ). This study also adds to the accumulating evidences for a broader role for LLPS in innate immunity. For example, in the cytoplasm, the DNA sensor cGAS forms spherical liquid condensa tes tha t are critical for its DN A sensing ca pacity ( 41 , 47 ). Here, we uncover the relevance of LLPS in the context of nuclear DNA sensing, thus further expanding the link between LLPS and the regulation of innate immunity. Of interest is also the different relationship between phosphorylation and LLPS in these contexts of cytoplasmic or nuclear sensing. While cGAS phosphorylation suppresses LLPS to pre v ent acti vation during mitosis ( 47 ), IFI16 phosphorylation within the IDR acts to promote LLPS and cytokine induction. This distinction highlights the di v ergent roles of phosphorylations in regulating LLPS. By extension, it is also possible for IFI16 to be inhibited through toggling between dif ferent phosphoryla tion sta tes during mitosis to pre v ent autoacti vation. Future studies can help determine whether the phosphorylation sites characterized in this study or other sites may also regulate IFI16 activity during mitosis.
Altogether, our study links phosphorylation-dri v en LLPS with nuclear DNA sensing. Our findings provide a mechanistic explanation for how IFI16 switch-like phase transitions, including dynamic oligomeric puncta and stable filamentation, are achieved with high temporal and spatial resolution for immune signaling initiation and maintenance. The IFI16 LLPS, as dictated by its phosphoryla tion sta tus, can serve as a biophysical rheosta t tha t toggles phases of innate immune activity. These results also pr omise to pr ovide valuable insights into the regulation of autoimmunity.

DA T A A V AILABILITY
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE ( 32 ) partner repository with the dataset identifier PXD034348 and 10.6019 / PXD034348. All PRM data has been deposited to Panorama Public ( 33 ): https:// panoramaw e b.org/IFI16 LLPS PRM.url . The RAW data and a compiled peptide library have been uploaded to Pro-teomeXchange (identifier PXD034348).